Taxanes are diterpene compounds which find utility in the pharmaceutical field. For example, paclitaxel (Taxol.RTM.), a taxane having the structure: ##STR1## has been found to be an effective anticancer agent.
Naturally occurring taxanes such as paclitaxel, 10-deacetylpaclitaxel and baccatin III can be extracted with some difficulty from the trunk bark of different species of Taxus (yew). Paclitaxel, in particular, may be extracted from the inner bark of Taxus brevifolia. Although T. brevifolia is a relatively common tree in the Pacific Northwest, it is a slow growing plant and is indigenous to the ecologically threatened old-growth forests of this area, and harvesting is thus increasingly restricted because of environmental concerns.
As yields of paclitaxel extracted from T. brevifolia are generally low, of the order of 100 mg/kg, semisynthetic methods of producing paclitaxel from baccatin III and 10-deacetylbaccatin have proven successful and are routinely practiced. Baccatin III, 10-deacetylbaccatin, as well as other paclitaxel precursors may be isolated from the needles of the European yew, Taxus baccata in relatively larger quantities, e.g. approximately 300 mg/kg of 10-deacetylbaccatin may be obtained from yew leaves. Although yew needles generally provide an adequate supply of the necessary starting materials for synthesizing paclitaxel, the supply is not endless and other methods easing the supply dilemma and producing adequate amounts of paclitaxel have become a priority. The art has thus continued to search for synthetic, including semisynthetic routes for the preparation of naturally occurring taxanes such as paclitaxel, as well as the preparation of paclitaxel analogues and second and third generation paclitaxel-like compounds thereof.
Recently, endophytic microbes associated with T. brevifolia were examined as potential alternative sources of paclitaxel. Stierle et al., in "Bioactive Metabolites of the Endophytic Fungi of Pacific Yew, Taxus brevifolia", ACS 1995, have confirmed that the fungus Taxomyces andreanae, isolated from the inner bark of a yew tree in Montana, has demonstrated the ability to produce paclitaxel.
Paclitaxel is converted to 6-.alpha.-hydroxy paclitaxel in human liver by cyp2C8, and loses most of its cytotoxicity as a result of this hydroxylation Such activity is evidenced by studies performed by Kumar et al, "Comparative in vitro Cytotoxic Effects of Paclitaxel and Its Major Human Metabolite 6.alpha.-hydroxypaclitaxel", Cancer Chemother. Pharmacol. 36: 129-135 (1995), and by Rahman et al., "Selective Biotransformation of Paclitaxel to 6-.alpha.-hydroxypaclitaxel by Human Cytochrome P450 2C8", Cancer Research, 54: 5543-5546 (1994). To avoid this problem, second generation analogs are being developed. Compounds such as 6-.alpha.-hydroxy-7-deoxytaxanes or compounds derived therefrom may be useful as second generation drugs. The chemical preparation of these compounds requires a long sequence of reactions to incorporate oxygen at C6. An enzyme able to hydroxylate the 6-position of a 7-deoxytaxane would afford a much simpler route. Although human cyp2C8 converts paclitaxel to 6-.alpha.-hydroxypaclitaxel, it has not been shown to be effective with 7-deoxypaclitaxel or 7-deoxybaccatin. We have, therefore, derived another enzyme source for this transformation.